Crocin Improves the Endothelial Function Regulated by Kca3.1 Through ERK and Akt Signaling Pathways

Based on the protective effect of crocin against cardiovascular diseases, we hypothesize that crocin could improve endothelial function through activating the eNOS(endothelial nitric oxide synthase) /NO pathway and/or the intermediate-conductance Ca2+-activated K+ channels (KCa3.1).

In this study, rat aortic rings were used to assess the regulatory effect of crocin on vascular tone and nitric oxide, prostacyclin, and KCa3.1, all endothelial vasodilators, were analyzed for effects by crocin. The expression profiles of p-eNOS, total-eNOS, p-ERK, total-ERK, p-Akt, total-Akt, KCa3.1, CD31, thrombomodulin, ICAM-1 and VCAM-1 were tested by western blotting. KCa3.1 was also analyzed by qPCR and immunofluorescence staining. Fluorescence and confocal microscopy were used to determine NO generation and intracellular Ca2+. Both EdU and MTT assays were used to evaluate cell viability. Cellular migration was assessed using transwell assay.

Crocin relaxed pre-contracted artery rings through either NO or KCa3.1, but not PGI, in an endothelium-dependent manner. Furthermore, crocin increased p-eNOS, total-eNOS expression and NO production as well as intracellular Ca2+ in both HUVECs and HUAECs (Human Umbilical Artery Endothelial cells). Crocin also stimulated the expression of CD31, thrombomodulin and vascular cell adhesion molecule 1 (VCAM-1), as well as increased cellular proliferation and migration in vitro. Interestingly, we determined for the first time that by blocking or silencing KCa3.1 there was inhibition of crocin induced upregulation of p-eNOS and total-eNOS. Correspondingly, the KCa3.1 inhibitor TRAM-34 also reduced the expression of CD31, thrombomodulin and VCAM-1, as well as diminished intracellular Ca2+, cellular proliferation and migration. Finally, crocin stimulated the expression of p-ERK, total-ERK, p-Akt and total-Akt, however suppression of MEK and Akt inhibited this expression profile in endothelial cells.

In the present study, these data strongly support the hypothesis that crocin could improve endothelial function through stimulation of the eNOS/NO pathway and other endothelial markers. This functional improvement is regulated by KCa3.1 via the MEK/ERK and PI3K/Akt signaling pathway.